Our work on theoretical, experimental, and computational aspects of light -tissue interactions for non-invasive quantitative optical imaging and spectroscopy has been continued. We continued our joint project with the PTB of Berlin and Politecnico di Milano aimed at quantification of optical properties of breast abnormalities (e.g., tumors), using time-domain scanning mammographs designed by the Berlin and Milan groups. Our analysis of their multi-spectral in vivo data, obtained at several projections, is based on the random walk model of time-resolved contrast functions. It resulted in estimates of the blood volume and oxygen saturation of the tumors and surrounding tissues. In most cases we were able to reconstruct also sizes of the abnormalities. Preliminary findings on the small sample of patients give indications that analyzed tumors (invasive ductal carcinoma) are hypoxic and have increased blood volume, comparing to surrounding normal tissue. We are pursuing the use of exogenous and endogenous fluorescent markers to be able to achieve specificity of the spectroscopic signatures of the tissue abnormality under investigation. In our animal experiments, we have devised an imaging system to quantify the concentration of fluorescinated antibodies binding to B or T tumor cells previously injected in the oral cavity of mice. Preliminary results show that our imaging system along with our theoretical algorithm are able to monitor non-invasively the concentration of fluorophore signals, as well as the pharmacokinetics associated with wash-out of the antibodies We are also continuing our study on using IR-dependent fluorescent detection methods to determine the position of sentinel node(s) to replace currently used detection by radioactive particles. We have extended our analysis of different phantom data and ex-vivo tissue to confirm the potential of the random walk-based theoretical approach to reconstruct with good accuracy 3D positions of deeply embedded fluorophores. In a series of animal experiments, we were successfully able to use near infrared nanoparticles to study their uptake of these particles through the blood circulation and in tumors. We were successfully able to image in real time these dynamics. We also have started a series of animal experiments. In these experiments, the tongues of Balb-C mice are injected with Squamous Carcinoma Cells that are CD3 and CD19 positive. FITCI conjugated antibodies to CD3 or CD19 are then injected into the tongue. Our goal is to study whether our model of diffuse fluorescent photon migration is able to separate the effects of light diffusion at a given depth from the actual distributions of the fluorescent antibodies. First, the tongue is imaged after injecting the fluorescent antibodies at different time intervals to assess the pharmacokinetics of the fluorescent antibodies. In a separate experiment, the same procedure is used, but the tongue is covered by slabs of agarose gel whose properties mimic that of human tissue. Two pertinent parameters, i.e., change in peak intensities of the fluorescent signals and their full width at the half maximum (FWHM) as a function of time are used to study the dynamics of labeled antibodies. As expected, results of the two experiments exhibit different functional form due to the ?degeneration? of the signal by tissue scattering . Using our model of diffuse fluorescent photon migration we are able to deconvolute the signal coming from deep structure and retrieve the true pharmacokinetics of tumor cells. These results show clearly that our mathematical model is able to quantify not only the concentration of fluorescently labeled antibodies, but their clearance and diffusion through tissue. We are investigating the use of three imaging modalities to quantify different parameters associated with blood circulation. These are: 1) thermography which provides a two-dimensional image of superficial skin temperatures. The concept is that higher temperatures occur in the skin superficial to veins that are involved in active transport of blood; 2) laser Doppler imaging LDI which produces two dimensional images of blood flow over a defined area at 690nm and 780nm; 3) multispectral imaging, designed in our Unit, which produces two dimensional images at six wavelengths (700-1000nm), by using a differential absorption algorithm (devised by our Unit) parameters related to the oxy- deoxy-hemoglobin ratio (i.e., oxygenation) and total blood content, as well as changes in other metabolic- and disease- related compounds such as cytochromes and ferritins can be derived. Two studies are underway: Kaposi?s sarcoma is a skin disease for which there is an NCI sponsored clinical trial evaluating the effectiveness of anti-angiogenetic drug treatment. Kaposi sarcoma (KS) is a highly vascular tumor. Angiogenesis and capillary permeability can play an important role in the development and progression of KS. Assessing responses to KS therapy is now generally performed by visually measuring and palpating the numerous lesions and using rather complex response criteria. No non-invasive standard technique is available to assess the effect of anti-angiogenetic therapy on blood flow in KS. The purpose of the clinical trial is to investigate the applicability of the three non-invasive methods for the assessment of vascularity and vascular changes associated with KS. Twenty patients have been investigated so far. Thermography, LDI and multispectral images were recorded over the lesion and compared to normal skin either adjacent to the lesion or on the contralateral side. Measurements were obtained prior to therapy and after receiving a regimen of liposomal doxorubicin and interleukin-12 for 18 weeks. Comparative image analysis between LDI and thermography in the present study showed a strong correlation between these two parameters The KS lesions generally had increased temperature and blood flux (as measured by LDI) as compared to normal skin. But interestingly observed that few patients had lower temperature in the lesion than normal. Similar results were also observed in LDI data. But the physiology behind this phenomenon was not clearly understood. The multispectral data show a large increase in blood volume, as well as intreging patterns of cytochrome concentration changes within the tumor. These patterns may yield information on the detailed effects of drugs on specific tumors. Further analysis of the data we are collecting promises to further our understanding of tumor angiogenesis and the effects of specific anti-angiogenetic treatment. The techniques are objective, easy to perform, and appear to be very sensitive in assessing improvement in the lesions upon administration of therapy. The approaches may thus have utility in monitoring trials of antiangiogenesis therapy in KS patients.